Trafficking of proteins to their appropriate destination is fundamental to the function of all living cells. This proposal explores aspects of the molecular mechanisms regulating intracellular distribution of thyroid hormone receptor (TR) subtypes. TRs are essential transcription factors that either activate or repress the expression of target genes in response to thyroid hormone (TH). TRs are encoded by two genes located on different chromosomes and produce a surprisingly diverse set of TRs through alternative splicing and internal initiation codons. Recent characterization of TR?1 isoforms with cytoplasmic functions adds a surprising twist to the intricacies of TR?s subcellular trafficking, and expands the diversity of the cellular response to TH. The proposed research explores our overarching hypothesis that mislocalization of TR contributes to pathogenesis in certain types of cancer and Resistance to Thyroid Hormone (RTH) syndrome. Our prior published studies supported by 2 R15 DK058028, and data from the current funding cycle provide a robust premise for the proposed research. Although they primarily reside in the nucleus, we have shown that TR?1 and TR?1 shuttle rapidly between the nucleus and cytoplasm. We have identified multiple nuclear localization and nuclear export signals within TR?1 and TR?1 that interact with importins and exportins, respectively, to mediate translocation into the nucleus. The fine balance between nuclear import and export of TRs has emerged as a critical control point for modulating TH-responsive gene expression, while an additional layer of complexity is added by multiple modular, often overlapping, functional domains. Here we will determine the structural interplay between nuclear, cytosolic, and mitochondrial targeting signals in the TRs. We will test our hypothesis that dominance of one targeting signal over another depends on key amino acid residues not only in the N-terminal A/B domain of TR but also in the ligand binding domain. GFP-tagged TR synthetic variants will be transfected into human cell lines and assayed for altered nuclear transport activity, sequestration in aggresomes, and mitochondrial localization by fluorescence microscopy. Given that TR contains multiple, conflicting targeting signals, the question arises of what factors are involved in nuclear retention. Here, we will determine whether RTH-associated mutations in TR?1 impact intranuclear mobility and alter interactions with NCoR1, MED1, and exportins, using a powerful combined approach of fluorescence recovery after photobleaching (FRAP), knockout or knockdown of MED1 and NCoR1, and coimmunoprecipitation assays. The proposed research investigates the complex interplay between TR?s dynamic transport pathways and TH signaling activities, and the correlation between altered trafficking signals and RTH. Our long-term aspiration is to provide a foundational springboard for the development of new therapeutic strategies for TR-associated disorders.